The Binding Sites of Cytochalasin D 11. THEIR RELATIONSHIP TO HEXOSE TRANSPORT AND TO CYTOCHALASIN B JANET TANNENBAUM,' STUART W. TANENBAUM AND GABRIEL C. GODMAN Departments of Microbiology and Pathology, Columbia Uniuersity, New York, New York 10032
ABSTRACT Cytochalasin B (CB) was able to compete with tritiated cytochalasin D (3H-CD)for binding sites in HEp-2 cells. The pattern of inhibition suggested that CB associates with a low affinity class of CD binding sites. Glucose and maltose did not inhibit binding of 3H-CD to isolated HEp-2 plasma membrane. Inhibition of hexose transport by CD was negligible, but CD did not block the potent inhibition of this transport by CB. These results indicate that CD does not bind to the high affinity CB receptor reportedly associated with the hexose transport system, and that this receptor cannot mediate the morphological effects of CD. Both CD and CB induced contraction-zeoisis in HEp-2 cells; CB was less potent than CD, and their effects appeared to be additive. It was concluded that the high affinity binding sites for CD and CB are different, but that these congeners share a low affinity site. Both high and low affinity sites for CD appear to mediate its morphological effects; only the low affinity class appears to be involved for CB. Possible identification of the common low affinity binding site as actomyosin (detailed in Tannenbaum et al., '77) is further discussed.
Cytochalasins A and B inhibit hexose transport in many cell lines (Mizel and Wilson, '72; Kletzien and Perdue, '73), whereas cytochalasin E is not inhibitory (Kletzien and Perdue, '75). Cytochalasin D is less active, or in some cases totally inactive, in affecting cellular permeability to sugars (Miranda et al., '74a; McDaniel et al., '75). It has been demonstrated that depletion of cellular glucose cannot in itself produce the characteristic effects of cytochalasins on cell morphology and contractility (Estensen and Plagemann, '72; Zigmond and Hirsch, '72; Yamada and Wessells, '73; Taylor and Wessells, '73; inter alia). However, the relationship between that binding site which apparently mediates the inhibition of hexose transport by CB (Lin et al., '74; Lin and Spudich, '74b) and those sites(s) responsible for the contractile effects of cytochalasins (Miranda et al., '74a,b; Godman et al., '75) is in J. CELL. PHYSIOL.,91: 239-248.
need of clarification. Functional and biochemical characterization of the binding sites of the cytochalasins is, therefore, important not only for understanding the mode of action of these compounds, but also for further delineating mechanisms of cellular contractility and some aspects of membrane operation. In this communication, we demonstrate that CD and CB share a common binding site in HEp-2 cells and membranes, but that this locus is distinct from the CB receptor which is related to hexose transport. The site common to CD and CB is a low affinity binding site for CD. Our data suggest that both high and low affinity sites for CD mediate the exReceived May 24, '76. Accepted Sept. 22, '76. ' Present address: Laboratories of Virology, St. Jude Children's Research Hospital, P. 0. Box 318, Memphis, Tennessee 38101, U. S. A. Present address: School of Biology, Chemistry and Ecology, SUNY College of Environmental Science and Forestry, Syracuse, N. Y. 13210, U.S. A.
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treme contractile response which appears to be the basis for the morphological effects of the drug (Miranda et al., '74a,b; Godman et al., '75), whereas, only low affinity CD receptors recognize CB. MATERIALS AND METHODS
HEp-2 cells were grown as monolayers on glass in Eagle's minimal essential medium supplemented with 10% newborn calf serum, 60 U/ml penicillin, and 60 pg/ ml streptomycin (growth medium obtained from Grand Island Biological Co.). The plasma membrane fraction was isolated on sucrose gradients (Atkinson and Summers, '71) the microsomal fraction was obtained as a fluffy band of material at the Tris-30% sucrose interface. Cytochalasin D (CD) was isolated from cultures of Zygosporium masonii (Tanenbaum, '71) and 3H-CD was obtained as previously described (Tannenbaum et al., '75).3Cytochalasin B (CB) was a gift from Doctor S. B. Carter of Imperial Chemical Industries. Stock solutions of cytochalasins were prepared in DMSO (1mgl ml) and diluted as required before use. HEp-2 cells planted at a density of 2.5 X lo5cells/2 ml/vial were grown overnight in glass scintillation vials before use. Competition between 3H-CDand unlabeled CB or CD for binding to HEp-2 monolayers was measured by incubating replicate monolayers of HEp-2 for 15 minutes at 37°C in vials with growth medium containing a constant concentration of DMSO, and the indicated concentrations of non-radioactive CB or CD plus 0.06 pg/ml 3H-CD or 0.52 pg/ml 3H-CD. The medium was then aspirated and the monolayers were washed twice with chilled Earle's balanced saline solution (EBSS) before determination of cell-bound 3H-CD . Background counts were determined by performing these same procedures on vials mock-planted with growth medium lacking cells. For determination of the effect of glucose or deoxyglucose on binding of 3H-CDto HEp2 monolayers, these were similarly treated, except that the 3H-CD solution was prepared in glucose-free growth medium with D-glucose (Fisher Chemical) or 2-deoxy-D-
glucose (DOG, Pierce Chemicals) added at concentrations between 0.10 and 100 mM. Chilled glucose-free EBSS was used for washing the monolayers. Transport of 14C-DOG (New England Nuclear) was measured by two techniques. Preliminary experiments employed the method described in (Miranda et al. '74a). HEp-2 cells were grown in Linbro FB-1624-TC plastic trays, planted at lo5 cells/ well. Cells were then rinsed three times with warm glucose-free growth medium, and incubated at 37°C for 15 or 30 minutes with glucose-free (bicarbonate-buffered) growth medium containing 14C-DOG(0.05 or 0.18 mM) with various concentrations of CD or CB in a constant totaI concentration of DMSO. Monolayers were washed twice with EBSS, trypsinized, and quantitatively transferred to scintillation vials for counting. In later experiments (table 2), HEp-2 cells grown in glass scintillation vials were washed twice with warm glucose-free growth medium and incubated at 37°C for 20 minutes with glucose-free(HEPESbuffered) growth medium containing 14CDOG (0.023 mM) plus various concentrations of CD and/or CB at a constant concentration of DMSO. Vials were then placed on ice, the test solutions were aspirated, and the monolayers were subsequently washed three times with chilled EBSS. For examination of zeiosis, monolayers of HEp-2 cells (2.5 X lo5 cells/2 ml/dish) were grown on sterile 484 mm2glass coverslips in 35 mm plastic petri dishes (Falcon) in 5% C02-95% air at 37°C for about 22 hours before assay. Growth medium was then replaced with medium containing CD and/or CB (or DMSO control solutions); cells were incubated for two hours at 37"C, and washed rapidly in EBSS. Such preparations were then fixed with 85% ethanol10% formalin-5% acetic acid and stained with azure-eosin. A centrifugation assay was employed to measure binding of 3H-CD to HEp-2 plasma membrane and microsomal frac'Some details ahout the preparation, characterization and purity of the cytochalasin D and the 3H-cytochalasinD employed in these studies are given in Tannenbaum et al., '77.
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BINDING SITES OF CYTOCHALASIN D. 11. TABLE 1
Effect of sugars on binding of 3H-CD to HEp-2 plasma membrane Bound W C D Percent of control
A. Sucrose-containing buffer Experiment no. Control (sucrose) Glucose Maltose
I 100 123 n.d.
I1 100 127 158
B. Sucrose-free buffer Experiment no. Control (sucrose) Glucose Maltose
I 100 105 111
I11 100 101 112
I1 100 100 91
Replicate pellets of HEp-2 plasma membrane were assayed within each experiment; % of control refers to total dprn 3H-CD bound to a membrane pellet in comparison with replicate control pellet. A. HEp-2 plasma membrane suspended in SMT was incubated five (I) or ten (11) minutes with the indicated sugar. ‘H-CD was added, and incubation continued an additional 15 minutes. Final concentration of each added sugar: 0.15M. B. HEp-2 plasma membrane suspended in 10 mM Tris-1 mM MgCI, (sucrose-free SMT) was incubated five (I) and (11) or 15 (111) minutes at 37°C with the indicated sugar. After addition of W C D , samples were treated as in A (see MATERIALS AND METHODS for details). Final concentration of each sugar added was: 0.1 M for I and 11, 0 15 M for 111. Membrane-hound ‘H-CD was determined by the standard procedure.
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tions. Cell fractions were suspended in 0.4 ml SMT (0.25 M sucrose-1 mM MgCI2-10 mM Tris HCl, pH 7.0) containing 0.52 pg/ ml 3H-CD plus either 12.5 pgiml CB (in 1.25% DMSO, final concentration) or 1.25% DMSO (control). These were incubated 15 minutes at 37”C, centrifuged (7,000 X g for ten minutes at 0-4°C) and the pellets were washed in chilled SMT. After suspension in distilled water, aliquots were withdrawn for determination of protein (Lowry et al., ’51) and tritium. To examine the effect of sugars on binding of 3H-CD,replicate samples of HEp-2 plasma membrane in SMT or in 10 mM Tris-1 mM MgC12,pH 7.0 (“sucrose-free buffer”) were preincubated with sucrose, glucose, or maltose for 5 to 15 minutes at 37°C before addition of 3H-CD (details in legend, table 1).Samples were assayed for bound drug as described above, except that pellets in “sucrose-free buffer” were washed with chilled Tris-MgC12containing a 0.1 M concentration of the appropriate sugar rather than with SMT. Radioactivity was determined in samples digested in Soluene-100 (Packard) and counted for 3H or I4Cin an Omnifluor-toluene (4 g/l) scintillation cocktail (New Eng-
land Nuclear). Efficiency of counting for 3H was determined by internal standardization. Estimation of the proportion of cellbound 3H-CD present at high or low affinity sites were performed by gra hical techniques (Tannenbaum et al., ’777 employing the binding curve reported in Tannenbaum et al. (’75). RESULTS
Binding sites Although they differ in potency, CD and CB produce generally similar cytologic effects (Miranda et al., ’74a; Wessells et al., ’71; Carter, ’72). It was, therefore, anticipated that the two congeners might employ identical binding sites for triggering their common action. To explore this possible relationship, the ability of CB to compete with 3H-CD for HEp-2 binding sites was tested. As shown in figure 1, CB was able to decrease the amount of 3H-CD bound to HEp-2 cells, but was a less effective competitor than CD itself. In the presence of 0.06 pglml 3H-CD (0.12 p M ) , the maximum inhibition of binding by CB was about 16%, representing displacement of some 700 dpm of 3H-CD from monolayers (fig. 1A). When the concentration of
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ABSTRACT Cytochalasin B (CB) was able to compete with tritiated cytochalasin D (3H-CD)for binding sites in HEp-2 cells. The pattern of inhibition suggested that CB associates with a low affinity class of CD binding sites. Glucose and maltose did not inhibit binding of 3H-CD to isolated HEp-2 plasma membrane. Inhibition of hexose transport by CD was negligible, but CD did not block the potent inhibition of this transport by CB. These results indicate that CD does not bind to the high affinity CB receptor reportedly associated with the hexose transport system, and that this receptor cannot mediate the morphological effects of CD. Both CD and CB induced contraction-zeoisis in HEp-2 cells; CB was less potent than CD, and their effects appeared to be additive. It was concluded that the high affinity binding sites for CD and CB are different, but that these congeners share a low affinity site. Both high and low affinity sites for CD appear to mediate its morphological effects; only the low affinity class appears to be involved for CB. Possible identification of the common low affinity binding site as actomyosin (detailed in Tannenbaum et al., '77) is further discussed.
Cytochalasins A and B inhibit hexose transport in many cell lines (Mizel and Wilson, '72; Kletzien and Perdue, '73), whereas cytochalasin E is not inhibitory (Kletzien and Perdue, '75). Cytochalasin D is less active, or in some cases totally inactive, in affecting cellular permeability to sugars (Miranda et al., '74a; McDaniel et al., '75). It has been demonstrated that depletion of cellular glucose cannot in itself produce the characteristic effects of cytochalasins on cell morphology and contractility (Estensen and Plagemann, '72; Zigmond and Hirsch, '72; Yamada and Wessells, '73; Taylor and Wessells, '73; inter alia). However, the relationship between that binding site which apparently mediates the inhibition of hexose transport by CB (Lin et al., '74; Lin and Spudich, '74b) and those sites(s) responsible for the contractile effects of cytochalasins (Miranda et al., '74a,b; Godman et al., '75) is in J. CELL. PHYSIOL.,91: 239-248.
need of clarification. Functional and biochemical characterization of the binding sites of the cytochalasins is, therefore, important not only for understanding the mode of action of these compounds, but also for further delineating mechanisms of cellular contractility and some aspects of membrane operation. In this communication, we demonstrate that CD and CB share a common binding site in HEp-2 cells and membranes, but that this locus is distinct from the CB receptor which is related to hexose transport. The site common to CD and CB is a low affinity binding site for CD. Our data suggest that both high and low affinity sites for CD mediate the exReceived May 24, '76. Accepted Sept. 22, '76. ' Present address: Laboratories of Virology, St. Jude Children's Research Hospital, P. 0. Box 318, Memphis, Tennessee 38101, U. S. A. Present address: School of Biology, Chemistry and Ecology, SUNY College of Environmental Science and Forestry, Syracuse, N. Y. 13210, U.S. A.
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treme contractile response which appears to be the basis for the morphological effects of the drug (Miranda et al., '74a,b; Godman et al., '75), whereas, only low affinity CD receptors recognize CB. MATERIALS AND METHODS
HEp-2 cells were grown as monolayers on glass in Eagle's minimal essential medium supplemented with 10% newborn calf serum, 60 U/ml penicillin, and 60 pg/ ml streptomycin (growth medium obtained from Grand Island Biological Co.). The plasma membrane fraction was isolated on sucrose gradients (Atkinson and Summers, '71) the microsomal fraction was obtained as a fluffy band of material at the Tris-30% sucrose interface. Cytochalasin D (CD) was isolated from cultures of Zygosporium masonii (Tanenbaum, '71) and 3H-CD was obtained as previously described (Tannenbaum et al., '75).3Cytochalasin B (CB) was a gift from Doctor S. B. Carter of Imperial Chemical Industries. Stock solutions of cytochalasins were prepared in DMSO (1mgl ml) and diluted as required before use. HEp-2 cells planted at a density of 2.5 X lo5cells/2 ml/vial were grown overnight in glass scintillation vials before use. Competition between 3H-CDand unlabeled CB or CD for binding to HEp-2 monolayers was measured by incubating replicate monolayers of HEp-2 for 15 minutes at 37°C in vials with growth medium containing a constant concentration of DMSO, and the indicated concentrations of non-radioactive CB or CD plus 0.06 pg/ml 3H-CD or 0.52 pg/ml 3H-CD. The medium was then aspirated and the monolayers were washed twice with chilled Earle's balanced saline solution (EBSS) before determination of cell-bound 3H-CD . Background counts were determined by performing these same procedures on vials mock-planted with growth medium lacking cells. For determination of the effect of glucose or deoxyglucose on binding of 3H-CDto HEp2 monolayers, these were similarly treated, except that the 3H-CD solution was prepared in glucose-free growth medium with D-glucose (Fisher Chemical) or 2-deoxy-D-
glucose (DOG, Pierce Chemicals) added at concentrations between 0.10 and 100 mM. Chilled glucose-free EBSS was used for washing the monolayers. Transport of 14C-DOG (New England Nuclear) was measured by two techniques. Preliminary experiments employed the method described in (Miranda et al. '74a). HEp-2 cells were grown in Linbro FB-1624-TC plastic trays, planted at lo5 cells/ well. Cells were then rinsed three times with warm glucose-free growth medium, and incubated at 37°C for 15 or 30 minutes with glucose-free (bicarbonate-buffered) growth medium containing 14C-DOG(0.05 or 0.18 mM) with various concentrations of CD or CB in a constant totaI concentration of DMSO. Monolayers were washed twice with EBSS, trypsinized, and quantitatively transferred to scintillation vials for counting. In later experiments (table 2), HEp-2 cells grown in glass scintillation vials were washed twice with warm glucose-free growth medium and incubated at 37°C for 20 minutes with glucose-free(HEPESbuffered) growth medium containing 14CDOG (0.023 mM) plus various concentrations of CD and/or CB at a constant concentration of DMSO. Vials were then placed on ice, the test solutions were aspirated, and the monolayers were subsequently washed three times with chilled EBSS. For examination of zeiosis, monolayers of HEp-2 cells (2.5 X lo5 cells/2 ml/dish) were grown on sterile 484 mm2glass coverslips in 35 mm plastic petri dishes (Falcon) in 5% C02-95% air at 37°C for about 22 hours before assay. Growth medium was then replaced with medium containing CD and/or CB (or DMSO control solutions); cells were incubated for two hours at 37"C, and washed rapidly in EBSS. Such preparations were then fixed with 85% ethanol10% formalin-5% acetic acid and stained with azure-eosin. A centrifugation assay was employed to measure binding of 3H-CD to HEp-2 plasma membrane and microsomal frac'Some details ahout the preparation, characterization and purity of the cytochalasin D and the 3H-cytochalasinD employed in these studies are given in Tannenbaum et al., '77.
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BINDING SITES OF CYTOCHALASIN D. 11. TABLE 1
Effect of sugars on binding of 3H-CD to HEp-2 plasma membrane Bound W C D Percent of control
A. Sucrose-containing buffer Experiment no. Control (sucrose) Glucose Maltose
I 100 123 n.d.
I1 100 127 158
B. Sucrose-free buffer Experiment no. Control (sucrose) Glucose Maltose
I 100 105 111
I11 100 101 112
I1 100 100 91
Replicate pellets of HEp-2 plasma membrane were assayed within each experiment; % of control refers to total dprn 3H-CD bound to a membrane pellet in comparison with replicate control pellet. A. HEp-2 plasma membrane suspended in SMT was incubated five (I) or ten (11) minutes with the indicated sugar. ‘H-CD was added, and incubation continued an additional 15 minutes. Final concentration of each added sugar: 0.15M. B. HEp-2 plasma membrane suspended in 10 mM Tris-1 mM MgCI, (sucrose-free SMT) was incubated five (I) and (11) or 15 (111) minutes at 37°C with the indicated sugar. After addition of W C D , samples were treated as in A (see MATERIALS AND METHODS for details). Final concentration of each sugar added was: 0.1 M for I and 11, 0 15 M for 111. Membrane-hound ‘H-CD was determined by the standard procedure.
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tions. Cell fractions were suspended in 0.4 ml SMT (0.25 M sucrose-1 mM MgCI2-10 mM Tris HCl, pH 7.0) containing 0.52 pg/ ml 3H-CD plus either 12.5 pgiml CB (in 1.25% DMSO, final concentration) or 1.25% DMSO (control). These were incubated 15 minutes at 37”C, centrifuged (7,000 X g for ten minutes at 0-4°C) and the pellets were washed in chilled SMT. After suspension in distilled water, aliquots were withdrawn for determination of protein (Lowry et al., ’51) and tritium. To examine the effect of sugars on binding of 3H-CD,replicate samples of HEp-2 plasma membrane in SMT or in 10 mM Tris-1 mM MgC12,pH 7.0 (“sucrose-free buffer”) were preincubated with sucrose, glucose, or maltose for 5 to 15 minutes at 37°C before addition of 3H-CD (details in legend, table 1).Samples were assayed for bound drug as described above, except that pellets in “sucrose-free buffer” were washed with chilled Tris-MgC12containing a 0.1 M concentration of the appropriate sugar rather than with SMT. Radioactivity was determined in samples digested in Soluene-100 (Packard) and counted for 3H or I4Cin an Omnifluor-toluene (4 g/l) scintillation cocktail (New Eng-
land Nuclear). Efficiency of counting for 3H was determined by internal standardization. Estimation of the proportion of cellbound 3H-CD present at high or low affinity sites were performed by gra hical techniques (Tannenbaum et al., ’777 employing the binding curve reported in Tannenbaum et al. (’75). RESULTS
Binding sites Although they differ in potency, CD and CB produce generally similar cytologic effects (Miranda et al., ’74a; Wessells et al., ’71; Carter, ’72). It was, therefore, anticipated that the two congeners might employ identical binding sites for triggering their common action. To explore this possible relationship, the ability of CB to compete with 3H-CD for HEp-2 binding sites was tested. As shown in figure 1, CB was able to decrease the amount of 3H-CD bound to HEp-2 cells, but was a less effective competitor than CD itself. In the presence of 0.06 pglml 3H-CD (0.12 p M ) , the maximum inhibition of binding by CB was about 16%, representing displacement of some 700 dpm of 3H-CD from monolayers (fig. 1A). When the concentration of
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